In the breakdown of heme, bile pigments, principally bilirubin, are produced in the serum which are then removed by the liver. The amount of bile pigments formed each day is closely related to the amount of hemoglobin destroyed and liver function. It is estimated that 1 gram (g) of hemoglobin yields 35 milligrams (mg) of bilirubin. Normally 0.1 to 1.5 mg of bilirubin is present in 100 milliliters (ml) of human plasma or serum.
Estimation of serum bilirubin has been recognized to be of great value in clinical studies, such as of liver dysfunction. A method for quantitatively assaying the bilirubin content of the serum was first devised by Van den Bergh by application of Ehrlich's test for bilirubin in urine. The Ehrlich reaction is based on the coupling of diazotized sulfanilic acid (Ehrlich's diazo reagent) and bilirubin to produce a reddish-purple azo compound. In the original procedure as described by Ehrlich, alcohol was used to provide a solution in which both bilirubin and the diazo reagent were soluble. Van den Bergh discovered that by omitting the alcohol when assaying for bile pigment in human bile normal development of the color occurred "directly", that is, without the addition of alcohol. This form of bilirubin which would react without the addition of alcohol was thus termed "direct-reacting." However, it was still necessary to add alcohol to detect bilirubin in normal serum. To that form of bilirubin which could be measured only after the addition of alcohol the term "indirect-reacting" was applied.
The indirect bilirubin is "free" (unconjugated) bilirubin en route to the liver from the reticuloendothelial tissues where the bilirubin is produced by the breakdown of heme porphyrins. Since this bilirubin is not water-soluble it requires addition of alcohol to initiate coupling with the diazo reagent. In the liver the free bilirubin becomes conjugated with glucuronic acid. Conjugated bilirubin, being water-soluble, can react directly with the diazo reagent so the the "direct bilirubin" of Van den Bergh is actually a bilirubin conjugate (bilirubin glucuronide).
Sulfonic acids other than sulfanilic acid have been suggested as acceptable in the diazo coupling reaction described. Such include p-toluenesulfonic acid, sulfosalicylic acid, sulfonic acid and hexamic acid. See, for example, U.S. Pat. No. 3,585,001.
It has also been known that other substances besides alcohol exhibit the same influence, that is of enhancing the diazo coupling of "free" bilirubin, allowing for a measure of indirect, and thus total, bilirubin. These substances are referred to as "accelerating agents" and have included caffeine, dyphylline, sodium acetate, sodium benzoate, gum arabic and others. Reference is made to Henry, R. J., Clinical Chemistry, Principles and Technics, Second Edition, Harper and Row, pp. 1047 (1974); With, T.K., Bile Pigments, Academic Press, pp. 324-327 (1968); and U.S. Pat. No. 4,038,031.
Test devices for bilirubin determination, such as in strip format, have been disclosed which make use of the diazo coupling reaction. See, for example, the above-identified patents as well as U.S. Pat. Nos. 3,853,476; 3,880,588; 3,912,457; 4,069,016; and 4,069,017. These devices have served a useful purpose in clinical diagnosis.
It has now been recognized, however, that these prior art devices suffer from the drawback that they do not absorb serum specimens in a uniform manner. The color formed at the point of sample application is quite intense, whereas very little, if any, color is developed peripheral to this point. This non-uniformity is a particularly undesirable characteristic, since uniform color development is necessary to achieve the precision required for a quantitative test.
This problem in prior art devices has, in accordance with the invention, been recognized and overcome as is fully described below.